RESEARCH PAPER Three aspartic acid residues of polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris are critical for inhibition of Fusarium phyllophilum PG F. Spinelli 1 , L. Mariotti 1 , B. Mattei 1 , G. Salvi 1 , F. Cervone 1 & C. Caprari 2 1 Dipartimento di Biologia Vegetale, Universita ` di Roma ‘‘La Sapienza’’, Rome, Italy 2 Dipartimento S.T.A.T. Universita ` del Molise, Contrada Fonte Lappone, Pesche (IS), Italy INTRODUCTION The perception of pathogens and the consequent activa- tion of plant defences is generally mediated by recogni- tion of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, peptidoglycans and proteins (Nu ¨rnberger et al. 2004). PAMPs are conserved among most pathogens and their perception triggers an immu- nity reaction called PTI (PAMP-triggered immunity) (Chisholm et al. 2006). Most phytopathogenic microorganisms produce enzymes that depolymerise the cell wall, the first barrier of the plant cell to an invading organism. Among cell wall degrading enzymes, PGs are secreted by pathogenic fungi at the early stages of infection and often cause tis- sue maceration by degrading homogalacturonan, the main component of pectin. PGs have been shown to be important virulence factors in interactions such as that of Botrytis cinerea and tomato or Claviceps purpurea and rye (ten Have et al. 1998; De Lorenzo & Ferrari 2002; Oeser et al. 2002). Proteins that inhibit fungal PGs (PGIPs) are present in the cell walls of many dicotyle- dons and monocotyledons (Federici et al. 2006). In some plants, the PGIP genes are organised in small families coding for closely related PGIP isoforms. In Phaseolus vulgaris, for instance, the inhibitor is encoded by four genes organised in two pairs, PvPGIP1 ⁄ PvPGIP2 and PvPGIP3 ⁄ PvPGIP4, which probably originated from independent events of gene duplication (D’Ovidio et al. 2004). PGIPs are thought to form bimolecular com- plexes (Caprari et al. 1996; Leckie et al. 1999; King et al. 2002; Kemp et al. 2004) with fungal PGs and favour the in vitro accumulation of oligogalacturonides, which are elicitors of the plant defence responses typically activated by PAMPs (De Lorenzo & Ferrari 2002; D’Ovidio et al. 2004; Federici et al. 2006). Interaction between PGs and PGIPs varies in terms of inhibition kinetics and strength, and reflects mutual adaptation between the enzymes and the inhibitors (Stotz et al. 2000; Federici et al. 2006). Keywords Pichia pastoris; polygalacturonase; polygalacturonase-inhibiting protein; site-directed mutagenesis. Correspondence C. Caprari, Dipartimento S.T.A.T. Universita ` del Molise, Contrada Fonte Lappone, I-86070 Pesche (IS), Italy. E-mail: claudio.caprari@unimol.it Editor C. Pieterse Received: 2 September 2008; Accepted: 31 October 2008 doi:10.1111/j.1438-8677.2008.00175.x ABSTRACT Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that specifically inhibit the activity of endopolygalacturonases (PGs) pro- duced by fungi during the infection process. The interaction with PGIPs limits the destructive potential of PGs and may trigger plant defence responses through the release of elicitor active oligogalacturonides. In order to pinpoint the residues of PvPGIP2 from Phaseolus vulgaris involved in the interaction with PGs, we used site-directed mutagenesis to mutate the resi- dues D131, D157 and D203, and tested for the inhibitory activity of the mutant proteins expressed in Pichia pastoris against Fusarium phyllophilum and Aspergillus niger PGs. Here, we report that mutation of these residues affects the inhibition capacity of PvPGIP2 against F. phyllophilum PG. Plant Biology ISSN 1435-8603 738 Plant Biology 11 (2009) 738–743 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands